For a cell to differentiate properly it must sense both the mechanical and chemical properties of its microenvironment. Sensing of the matrix mechanical properties plays a critical role and influences many aspects of cellular differentiation, regeneration and disease. Matrix mechanosensing is not understood in part because many of the steps involved occur at a molecular scale of nanometers. We have recently shown that submicrometer pillars are displaced at early times by a local contraction of 120 nm. This is not observed with larger (>1 mm) pillars. Because these early nanometer displacements are altered in cells that are unable to respond to matrix rigidity changes, we suggest that the cells use the force needed to produce a 120 nm displacement of matrix as a measure of matrix rigidity as others also believe. To understand how this process occurs will require a combination of submicrometer fabrication and quantitative cell biological techniques that the two investigators can bring to bear upon this problem. We will define the steps in the local contractions at a second and nanometer level using new nanofabricated devices that will test the effects of changes in matrix area, spacing and rigidity even on single cells. From a description of the steps in the process, we will analyze which molecular complexes are involved in each step. These same tools will enable us to analyze the mechanical factors that affect adhesion maturation and to know if local contractions continue in fully spread cells. Using mesenchymal stem cells, we will determine the time course of the contractions that precede differentiation in response to rigid and soft small pillars. This grant will enable us to determine the bases for the mechanochemical steps involved in stem cell differentiation that will enable targeting of the specific steps for manipulations of differentiation or disease processes. PUBLIC HEALTH RELEVANCE: The purpose of the proposed work is to understand the molecular basis of newly- discovered local contractions and their role in cellular rigidity sensing. We will employ arrays of elastomer pillars with sub-micron dimensions for force measurements; examine how these local contractions are modified in cells with modified ability to sense rigidity; and study long-term differentiation in mesenchymal stem cells.